Directed Enrichment of Genomic DNA for High-Throughput Sequencing

Abstract

The present invention provides microarrays of oligonucleotide primer pairs, and in particular, microarrays of primers that comprise at least one cleavable linkage. Also provided are methods to capture oligonucleotide primer pairs from one or more microarrays, and methods to use the captured oligonucleotide primer pairs, such as for amplification of a target polynucleotide sequence. In addition, methods of using a microarray to isolate, purify and/or amplify a target polynucleotide are provided.

1 - 39 . (canceled) 
     
     
         40 . A method for amplifying a target nucleic acid, comprising:
 a) providing a plurality of oligonucleotides having at least one cleavable linkage, wherein the plurality of oligonucleotides are immobilized to a support;   b) hybridizing a target nucleic acid to at least one immobilized oligonucleotide so as to produce a target/oligonucleotide complex;   c) embedding the target/oligonucleotide complex into a capturing support;   d) cleaving the at least one cleavable linkage in the immobilized oligonucleotide, so as to release the duplex into the capturing support; and   e) amplifying the target nucleic acid of the released duplex so as to produce an amplified target nucleic acid.   
     
     
         41 . The method of  claim 40 , wherein the capturing support in step (c) comprises a plurality of primer oligonucleotides which hybridize to the target nucleic acid. 
     
     
         42 . The method of  claim 40 , wherein the target nucleic acid comprises a genomic DNA fragment. 
     
     
         43 . The method of  claim 40 , wherein the capturing support is a polyacrylamide gel. 
     
     
         44 . The method of  claim 40 , wherein the polyacrylamide gel is a reversible polyacrylamide gel. 
     
     
         45 . A method for amplifying a target nucleic acid, comprising:
 a) providing a plurality of oligonucleotides having at least one cleavable linkage, wherein the plurality of oligonucleotides are immobilized to a support;   b) hybridizing a target nucleic acid to at least one immobilized oligonucleotide so as to produce a target/oligonucleotide complex, wherein each end of the target nucleic acid is ligated to a universal adapter;   c) conducting a primer extension reaction on the target/oligonucleotide complex so as to produce a primer extension product;   d) removing the target nucleic acid from the primer extension product so as to leave the primer extension product immobilized to the support;   e) embedding the immobilized primer extension product into a capturing support; and   f) amplifying the immobilized primer extension product so as to produce a plurality of amplification products in the capturing support.   
     
     
         46 . The method of  claim 45 , wherein the capturing support of step (e) comprises at least one primer oligonucleotide that hybridizes to the immobilized primer extension product. 
     
     
         47 . The method of  claim 45  further comprising cleaving the at least one cleavable linkage in the embedded immobilized primer extension product before step (f), so as to release the primer extension product into the capturing support. 
     
     
         48 . The method of  claim 45  further comprising cleaving the embedded immobilized primer extension product after step (f) so as to release the plurality of amplification products into the capturing support. 
     
     
         49 . The method of  claim 45 , wherein the target nucleic acid comprises a genomic DNA fragment. 
     
     
         50 . The method of  claim 45 , wherein the capturing support is a polyacrylamide gel. 
     
     
         51 . The method of  claim 45 , wherein the polyacrylamide gel is a reversible polyacrylamide gel. 
     
     
         52 . A method for amplifying a target nucleic acid, comprising:
 a) providing a plurality of oligonucleotides having at least one cleavable linkage, wherein the plurality of oligonucleotides are immobilized to a support;   b) hybridizing a target nucleic acid to at least one immobilized oligonucleotide so as to produce an immobilized target/oligonucleotide complex, wherein each end of the target nucleic acid is ligated to a universal adapter;   c) conducting a primer extension reaction on the target/oligonucleotide complex so as to produce an immobilized primer extension product;   d) embedding the immobilized primer extension product into a capture support;   e) cleaving the at least one cleavable linkage so as to release the primer extension product into the capturing support;   f) removing the target nucleic acid from the primer extension product; and   g) amplifying the immobilized primer extension product so as to produce a plurality of amplification products in the capturing support.   
     
     
         53 . The method of  claim 52 , wherein the capturing support of step (d) comprises at least one primer oligonucleotide that hybridizes to the primer extension product. 
     
     
         54 . The method of  claim 52 , wherein the target nucleic acid comprises a genomic DNA fragment. 
     
     
         55 . The method of  claim 52 , wherein the capturing support is a polyacrylamide gel. 
     
     
         56 . The method of  claim 52 , wherein the polyacrylamide gel is a reversible polyacrylamide gel.